Journal: Life Science Alliance
Article Title: Apolipoprotein E intersects with amyloid-β within neurons
doi: 10.26508/lsa.202201887
Figure Lengend Snippet: (A) Schematic overview of ApoE KO, ApoE3 KI, and ApoE4 KI astrocyte-conditioned medium collection and subsequent 4 h treatment of ApoE KO primary neuron cultures (19 DIV). (B, C) Representative fluorescent images obtained by epifluorescence microscopy of ApoE KO neurons treated with ApoE3 or ApoE4 astrocyte-conditioned medium for 4 h with the focus on neuronal cell bodies. The ApoE astrocyte medium-treated neurons were labeled for ApoE (green), neuronal dendrite marker MAP2 (grey), and either LAMP1 (B) or Rab7 (C) (both shown in magenta). The left panels (B, C) show an overview of the entire neuron; higher magnification images are shown in the other four panels. The white arrows indicate ApoE puncta overlap with LAMP1-positive vesicles in neuronal cell bodies (B). Left panels: scale bars are 40 μm. Right panels: scale bars are 10 μm. (D, E, F, G) Representative epifluorescence images of neurites from ApoE3 and ApoE4 media-treated primary neurons. (D, E, F, G) The neurites are labeled with ApoE (green), MAP2 (D, E) (grey), and late endosomal/lysosomal marker LAMP1 (D), late endosomal marker Rab7 (E), autophagosome marker LC3β (F) or early endosomal marker EEA1 (G) (all in magenta). (D, E, F, G) White arrows indicate co-localization between ApoE and the subcellular markers (D, E, F, G). Scale bars represent 10 μm. (H) Schematic representation of the lysosome degradation inhibitor Baf A1 and astrocytic ApoE treatment of ApoE KO primary neurons (19 DIV). (H, I, J) Representative fluorescence images of ApoE KO neurites treated with control (DMSO) or 10 nM lysosomal inhibitor bafilomycin A1 for 1 h, followed by 4 h ApoE3 (H) or ApoE4 (I) astrocyte-conditioned media. The neurites were labeled for ApoE (green), LAMP1 (red) and MAP2 (grey). ApoE, and LAMP1 co-localization, indicating the presence of ApoE at late endosomes and/or autophagosomes, is indicated by white arrows. Scale bar is 15 μm. (K, L) Quantification of ApoE and LAMP1 co-localization in ApoE3-treated neurons shown in (K) and ApoE4-treated neurons shown in (L) with and without bafilomycin A1 treatment. The researcher performing the quantifications was blinded. The percentages of ApoE co-localizing to LAMP1-positive pixels increased from 5.2–9.8% (Mann–Whitney test, P = 0.0864) in ApoE3-treated and decreased from 8.7–6.3% (Mann–Whitney test, P = 0.4358) in ApoE4-treated neurons after Baf A1 treatment (compared with DMSO control), although these changes were not statistically significant; number of cultures: 3; number of neurons analyzed within each culture: 7; for each neuron, 10 neurites were analyzed and averaged. (M) Quantification of the puncta size of added astrocytic ApoE in neurites with and without Baf A1 treatment. No significant difference in ApoE puncta size was observed after Baf A1 treatment in ApoE3- and ApoE4-treated neurons (mean ApoE puncta size: ApoE3 + DMSO: 10.3 pixels, ApoE3 + Baf A1: 9.0 pixels, ApoE4 + DMSO: 9.6 pixels, and ApoE4 + Baf A1: 9.4 pixels) (Kruskal–Wallis test, P = 0.7064); number of cultures: 3; number of neurons analyzed within each culture: 6–7; for each neuron, 10 neurites were analyzed and averaged. Data are shown as mean ± SD. (N) Representative fluorescence images showing human ApoE (green) are localized at or close to dendritic spines (indicated by a white arrow) labeled by phalloidin (magenta) in neurites treated with ApoE3 astrocyte-conditioned media for 4 h. Scale bar is 6 μm. See also .
Article Snippet: Rabbit polyclonal anti-early endosomal antigen 1 (EEA1) , Sigma-Aldrich , Cat# E4156; RRID: AB_609870.
Techniques: Epifluorescence Microscopy, Labeling, Marker, Fluorescence, MANN-WHITNEY
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